The Dynamics of Golgi Protein Traffic Visualized in Living Yeast Cells

We describe for the first time the visualization of Golgi membranes in living yeast cells, using green fluorescent protein (GFP) chimeras. Late and early Golgi markers are present in distinct sets of scattered, moving cisternae. The immediate effects of temperature-sensitive mutations on the distribution of these markers give clues to the transport processes occurring. We show that the late Golgi marker GFP-Sft2p and the glycosyltransferases, Anp1p and Mnn1p, disperse into vesicle-like structures within minutes of a temperature shift in sec18, sft1, and sed5 cells, but not in sec14 cells. This is consistent with retrograde vesicular traffic, mediated by the vesicle SNARE Sft1p, to early cisternae containing the target SNARE Sed5p. Strikingly, Sed5p itself moves rapidly to the endoplasmic reticulum (ER) in sec12 cells, implying that it cycles through the ER. Electron microscopy shows that Golgi membranes vesiculate in sec18 cells within 10 min of a temperature shift. These results emphasize the dynamic nature of Golgi cisternae and satisfy the kinetic requirements of a cisternal maturation model in which all resident proteins must undergo retrograde vesicular transport, either within the Golgi complex or from there to the ER, as anterograde cargo advances.

Radial and longitudinal diffusion of myoglobin in single living heart and skeletal muscle cells

We have used a fluorescence recovery after photobleaching (FRAP) technique to measure radial diffusion of myoglobin and other proteins in single skeletal and cardiac muscle cells. We compare the radial diffusivities, Dr (i.e., diffusion perpendicular to the long fiber axis), with longitudinal ones, Dl (i.e., parallel to the long fiber axis), both measured by the same technique, for myoglobin (17 kDa), lactalbumin (14 kDa), and ovalbumin (45 kDa). At 22°C, Dl for myoglobin is 1.2 × 10−7 cm2/s in soleus fibers and 1.1 × 10−7 cm2/s in cardiomyocytes. Dl for lactalbumin is similar in both cell types. Dr for myoglobin is 1.2 × 10−7 cm2/s in soleus fibers and 1.1 × 10−7 cm2/s in cardiomyocytes and, again, similar for lactalbumin. Dl and Dr for ovalbumin are 0.5 × 10−7 cm2/s. In the case of myoglobin, both Dl and Dr at 37°C are about 80% higher than at 22°C. We conclude that intracellular diffusivity of myoglobin and other proteins (i) is very low in striated muscle cells, ≈1/10 of the value in dilute protein solution, (ii) is not markedly different in longitudinal and radial direction, and (iii) is identical in heart and skeletal muscle. A Krogh cylinder model calculation holding for steady-state tissue oxygenation predicts that, based on these myoglobin diffusivities, myoglobin-facilitated oxygen diffusion contributes 4% to the overall intracellular oxygen transport of maximally exercising skeletal muscle and less than 2% to that of heart under conditions of high work load.

Phosphatases in the cell surface of living yeast cells /

Work performed at the University of Rochester.

Application of Mesenchymal Stem Cells for repair and regeneration of cartilage and bone

Australian efforts to provide orthopaedic surgeons with living, load-bearing scaffolds suitable for current joint (knee and hip) replacement surgery, non-union fracture repair, and miniscal and growth plate cartilage regeneration are being lead by teams at the Institute for Medical and Veterinary Science and Women's and Children's Hospital in Adelaide; the Peter MacCallum and St Vincent's Medical Research Institutes in Melbourne; and the Mater Medical Research Institute and new Institute for Health and Biomedical Innovation at QUT, Brisbane. In each case multidisciplinary teams are attempting to develop autologous living tissue constructs, utilising mesenchymal stem cells (MSC), with the intention of effecting seamless repair and regeneration of skeletal trauma and defects. In this article we will briefly review current knowledge of the phenotypic properties of MSC and discuss the potential therapeutic applications of these cells as exemplified by their use in cartilage repair and tissue engineering based approaches to the treatment of skeletal defects.

Polymer nanocarrier system for endosome escape and timed release of siRNA with complete gene silencing and cell death in cancer cells

An influenza virus-inspired polymer mimic nanocarrier was used to deliver siRNA for specific and near complete gene knockdown of an osteoscarcom cell line (U-2SO). The polymer was synthesized by single-electron transfer living radical polymerization (SET-LRP) at room temperature to avoid complexities of transfer to monomer or polymer. It was the only LRP method that allowed good block copolymer formation with a narrow molecular weight distribution. At nitrogen to phosphorus (N/P) ratios of equal to or greater than 20 (greater than a polymer concentration of 13.8 μg/mL) with polo-like kinase 1 (PLK1) siRNA gave specific and near complete (>98%) cell death. The polymer further degrades to a benign polymer that showed no toxicity even at polymer concentrations of 200 μg/mL (or N/P ratio of 300), suggesting that our polymer nanocarrier can be used as a very effective siRNA delivery system and in a multiple dose administration. This work demonstrates that with a well-designed delivery device, siRNA can specifically kill cells without the inclusion of an additional clinically used highly toxic cochemotherapeutic agent. Our work also showed that this excellent delivery is sensitive for the study of off-target knockdown of siRNA.

Genome sequencing of cells that live inside glass cages reveals their past history

One-quarter of the total primary production on earth is contributed by diatoms1. These are photosynthetic, unicellular algae with ornamented silica shells found in all aquatic and moist environments. They form the base of energy-efficient food webs that support all aquatic life forms. More than 250 genera of living diatoms, with as many as 100,000 species are known2. Fossil diatoms are known as early as the Cretaceous, 144–65 m.y. ago3. In India, deposits of diatoms occur in Rajasthan and are known as ‘multani mitti’. Multani mitti or Indian Fuller’s earth or diatomaceous earth as it is called in the West, is applied as a paste on the surface of the skin for 15–20 min and then washed-off. This leaves the skin feeling smooth, soft, moist and rejuvenated. Diatomaceous earth is now being used in the formulation of soaps, cleansing products, face powders and skincare preparations. Diatomaceous earth is a mineral material consisting mainly of siliceous fragments of various species of fossilized remains of diatoms.

A metabolomic approach to studying mechanisms of polymeric gene delivery to retinal pigment epithelial cells

Tiivistelmä ReferatAbstract Metabolomics is a rapidly growing research field that studies the response of biological systems to environmental factors, disease states and genetic modifications. It aims at measuring the complete set of endogenous metabolites, i.e. the metabolome, in a biological sample such as plasma or cells. Because metabolites are the intermediates and end products of biochemical reactions, metabolite compositions and metabolite levels in biological samples can provide a wealth of information on on-going processes in a living system. Due to the complexity of the metabolome, metabolomic analysis poses a challenge to analytical chemistry. Adequate sample preparation is critical to accurate and reproducible analysis, and the analytical techniques must have high resolution and sensitivity to allow detection of as many metabolites as possible. Furthermore, as the information contained in the metabolome is immense, the data set collected from metabolomic studies is very large. In order to extract the relevant information from such large data sets, efficient data processing and multivariate data analysis methods are needed. In the research presented in this thesis, metabolomics was used to study mechanisms of polymeric gene delivery to retinal pigment epithelial (RPE) cells. The aim of the study was to detect differences in metabolomic fingerprints between transfected cells and non-transfected controls, and thereafter to identify metabolites responsible for the discrimination. The plasmid pCMV-β was introduced into RPE cells using the vector polyethyleneimine (PEI). The samples were analyzed using high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC) coupled to a triple quadrupole (QqQ) mass spectrometer (MS). The software MZmine was used for raw data processing and principal component analysis (PCA) was used in statistical data analysis. The results revealed differences in metabolomic fingerprints between transfected cells and non-transfected controls. However, reliable fingerprinting data could not be obtained because of low analysis repeatability. Therefore, no attempts were made to identify metabolites responsible for discrimination between sample groups. Repeatability and accuracy of analyses can be influenced by protocol optimization. However, in this study, optimization of analytical methods was hindered by the very small number of samples available for analysis. In conclusion, this study demonstrates that obtaining reliable fingerprinting data is technically demanding, and the protocols need to be thoroughly optimized in order to approach the goals of gaining information on mechanisms of gene delivery.

Study of the effect of alcohol on single human red blood cells using near-infrared laser tweezers Raman spectroscopy

The effect of alcohol solution on single human red blood Cells (RBCs) was investigated using near-infrared laser tweezers Raman spectroscopy (LTRS). In our system, a low-power diode laser at 785 nm was applied for the trapping of a living cell and the excitation of its Raman spectrum. Such a design could simultaneously reduce the photo-damage to the cell and suppress the interference from the fluorescence on the Raman signal. The denaturation process of single RBCs in 20% alcohol solution was investigated by detecting the time evolution of the Raman spectra at the single-cell level. The vitality of RBCs was characterized by the Raman band at 752 cm(-1), which corresponds to the porphyrin breathing mode. We found that the intensity of this band decreased by 34.1% over a period of 25 min after the administration of alcohol. In a further study of the dependence of denaturation on alcohol concentration, we discovered that the decrease in the intensity of the 752 cm(-1) band became more rapid and more prominent as the alcohol concentration increased. The present LTRS technique may have several potential applications in cell biology and medicine, including probing dynamic cellular processes at the single cell level and diagnosing cell disorders in real time. Copyright (c) 2005 John Wiley T Sons, Ltd.

Mechanisms of Transposable Element Repression by Piwi Proteins in the piRNA Pathway of Drosophila Germ Cells

The ability to reproduce is a defining characteristic of all living organisms. During reproduction, the integrity of genetic material transferred from one generation to the next is of utmost importance. Organisms have diverse strategies to ensure the fidelity of genomic information inherited between generations of individuals. In sexually reproducing animals, the piRNA pathway is an RNA-interference (RNAi) mechanism that protects the genomes of germ cells from the replication of ‘selfish’ genetic sequences called transposable elements (TE). When left unabated, the replication of TE sequences can cause gene disruption, double-stranded DNA breaks, and germ cell death that results in sterility of the organism. In Drosophila, the piRNA pathway is divided into a cytoplasmic and nuclear branch that involves the functions of three Piwi-clade Argonaute proteins—Piwi, Aubergine (Aub) and Argonaute-3 (Ago3)—which bind piwi-interacting RNA (piRNA) to form the effector complexes that represses deleterious TE sequences.

The work presented in this thesis examines the function and regulation of Piwi proteins in Drosophila germ cells. Chapter 1 presents an introduction to piRNA biogenesis and to the essential roles occupied by each Piwi protein in the repression of TE. We discuss the architecture and function of germ granules as the cellular compartments where much of the piRNA pathway operates. In Chapter 2, we present how Piwi in the nucleus co-transcriptionally targets genomic loci expressing TE sequences to direct the deposition of repressive chromatin marks. Chapter 3 examines the cytoplasmic function of the piRNA pathway, where we find that the protein Krimper coordinates Aub and Ago3 in the piRNA ping-pong pathway to adaptively target and destroy TE transcripts. Chapter 4 explores how interactions of Piwis with associated proteins are modulated by arginine methylation modifications. Lastly, in Chapter 5 I present evidence that the cytoplasmic branch of the piRNA pathway can potentially ‘cross-talk’ with the nuclear branch to transfer sequence information to better target and co-transcriptionally silence the genomic loci coding active TE sequences. Overall, the work presented in this thesis constitutes a part of the first steps in understanding the molecular mechanisms that protect germ cells from invasion by TE sequences.

Identification and characterization of hypoxia-induced genes in Carassius auratus blastulae embryonic cells using suppression subtractive hybridization

Organisms living in water are inevitably exposed to periods of hypoxia. Environmental hypoxia has been an important stressor having manifold effects on aquatic life. Many fish species have evolved behavioral, physiological, biochemical and molecular adaptations that enable them to cope with hypoxia. However, the molecular mechanisms of hypoxia tolerance in fish, remain unknown. in this study, we used suppression subtractive hybridization to examine the differential gene expression in CAB cells (Carassius auratus blastulae embryonic cells) exposed to hypoxia for 24 h. We isolated 2100 clones and identified 211 differentially expressed genes (e-value <= 5e-3; Identity > 45%). Among the genes whose expression is modified in cells, a vast majority involved in metabolism, signal transduction, cell defense, angiogenesis, cell growth and proliferation. Twelve genes encoding for ERO1-L, p53, CPO, HO-1, MKP2, PFK-2, cystatin B, GLUT1, BTG1, TGF beta 1, PGAM1, hypothetical protein F1508, were selected and identified to be hypoxia-induced using semi-quantitive RT-PCR and real-time PCR. Among the identified genes, two open reading frames (ORFs) encoding for CaBTG1 and Cacystatin B were obtained. The deduced amino acid sequence of CaBTG1 had 94.1%, 72.8%, 72.8%, 72.8%, 68.6% identity with that of DrBTG1, HsBTG1, BtBTG1, MmBTG1 and XIBTG1. Comparison of Cacystatin B with known cystatin B, the molecules exhibited 49.5 to 76.0% identity overall. These results may provide significant information for further understanding of the adaptive mechanism by which C. auratus responds to hypoxia. (c) 2008 Elsevier Inc. All rights reserved.

Organic-inorganic hybrid material for the cells immobilization: Long-term viability mechanism and application in BOD sensors

In this paper, organic-inorganic hybrid material, which is composed of silica and the grafting copolymer of poly (vinyl alcohol) and 4-vinylpyridine (PVA-g-P(4-VP)), was employed to immobilize Trichosporon cutaneum strain 2.570 cells. Cells entrapped into the hybrid material were found to keep a long-term viability. The mechanism of such a long-term viability was investigated by using confocal laser scanning microscopy (CLSM). Our studies revealed that arthroconidia produced in the extracellular material might play an important role in keeping the long-term viability of the immobilized microorganism. After the arthroconidia were activated, an electrochemical biochemical oxygen demand (BOD) sensor based on cell/hybrid material-modified supporting membrane was constructed for verifying the proposed mechanism.

Functional gold nanoparticles for studying the interaction of lectin with glycosyl complex on living cellular surfaces

In this study. lectin-conjugated gold nanoparticles (GNPs) were prepared by standard biotin-streptavidin chemistry. The lectin-conjugated GNPs call be used as ail indicator for studying the interaction of lectin with glycosyl complex on living cellular Surfaces due to the high affinity of the lectin with saccharides. The interactions of two well-known lectins (Ricinus communis agglutinin and concanavalin A) and three different cell lines (HeLa, 293, and 293T) were selected here to establish this assay. Highly binding affinity of R. communis agglutinin with cells was demonstrated by conventional microscopic and UV-visible spectroscopic Studies. In addition, the binding process can be inhibited by galactose, giving further proof of the binding mechanism. (c) 2009 Elsevier Inc. All rights reserved.

Purification of human tissue prokallikrein excreted from insect cells by liquid chromatography

Tissue kallikrein, generally existing in living bodies as prokallikrein, is a serine proteinase that has proven of great significance to treat hypertension, cardiopathy and nephropathy. Although the extraction of tissue kallikrein from human urine is the most commonly used method to obtain such a protein, not only the yield is very little, but also the procedure is rather complex. Furthermore, the biological safety is uncertain. Therefore, the preparation of such a protein by genetic engineering method, including gene expression, cell culture, separation and purification, is very important. In this paper, a new method to obtain purified tissue prokallikrein excreted from insect cells by liquid chromatography has been proposed. In contrast to the previously published papers, the purification procedure is simplified to only three steps with the final yield of 57% and the purity of 95%, which is not only convenient, but also low-cost and suitable for the large-scale preparation of such a protein. The purified protein is further validated as prokallikrein by high performance liquid chromatography-mass spectrometry and amino acid sequencing. (c) 2005 Elsevier B.V. All rights reserved.

Protection of cells from salinity stress by extracellular polymeric substances in diatom biofilms

Diatom biofilms are abundant in the marine environment. It is assumed (but untested) that extracellular polymeric substances(EPS), produced by diatoms, enable cells to cope with fluctuating salinity. To determine the protective role of EPS, Cylindrotheca closterium was grown in xanthan gum at salinities of 35, 50, 70 and 90 ppt. A xanthan matrix significantly increased cell viability (determined by SYTOX-Green), growth rate and population density by up to 300, 2, 300 and 200%, respectively. Diatoms grown in 0.75% w/v xanthan, subjected to acute salinity shock treatments (at salinities 17.5, 50, 70 and 90 ppt) maintained photosynthetic capacity, F_q′/F_m′, within 4% of pre-shock values, whereas F_q′/F_m′ in cells grown without xanthan declined by up to 64% with hypersaline shock. Biofilms that developed in xanthan at standard salinity helped cells to maintain function during salinity shock. These results provide evidence of the benefits of living in an EPS matrix for biofilm diatoms.

Quantification of nanoparticle uptake by cells using microscopical and analytical techniques

Quantification of nanoparticles in biological systems (i.e., cells, tissues and organs) is becoming a vital part of nanotoxicological and nanomedical fields. Dose is a key parameter when assessing behavior and any potential risk of nanomaterials. Various techniques for nanoparticle quantification in cells and tissues already exist but will need further development in order to make measurements reliable, reproducible and intercomparable between different techniques. Microscopy allows detection and location of nanoparticles in cells and has been used extensively in recent years to characterize nanoparticles and their pathways in living systems. Besides microscopical techniques (light microscopy and electron microscopy mainly), analytical techniques such as mass spectrometry, an established technique in trace element analysis, have been used in nanoparticle research. Other techniques require 'labeled particles, fluorescently, radioactively or magnetically. However, these techniques lack spatial resolution and subcellular localization is not possible. To date, only electron microscopy offers the resolving power to determine accumulation of nanoparticles in cells due to its ability to image particles individually. So-called super-resolution light microscopy techniques are emerging to provide sufficient resolution on the light microscopy level to image or 'see particles as individual particles. Nevertheless, all microscopy techniques require statistically sound sampling strategies in order to provide quantitative results. Stereology is a well-known sampling technique in various areas and, in combination with electron microscopy, proves highly successful with regard to quantification of nanoparticle uptake by cells. © 2010 Future Medicine Ltd.